Peptide derived from gpc3, pharmaceutical composition for treatment or prevention of cancer using same, immunity inducer, and method for producing antigen-presenting cells

ABSTRACT

The present invention provides a peptide containing 8 or more consecutive amino acid residues in an amino acid sequence of any of SEQ ID NOS: 1 to 11 and consisting of 11 or less amino acid residues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/185,734, filed Nov. 9, 2018, which is a Divisional of U.S.application Ser. No. 15/556,694, which is the U.S. National Stageapplication of PCT/JP2016/057356, filed Mar. 9, 2016, which claimspriority from Japanese application JP 2015-046463, filed Mar. 9, 2015.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 31, 2020, isnamed sequence.txt and is 8,100 bytes.

TECHNICAL FIELD

The present invention relates to a GPC3-derived peptide, morespecifically an immunogenic peptide for presenting an antigen to a Tcell via binding to a human leukocyte antigen, a pharmaceuticalcomposition for treating or preventing cancer using the same, animmunity inducer, a method for producing an antigen-presenting cell, andthe like.

BACKGROUND ART

Although it is considered that cancer cells always incidentally appearin a living body, it is hypothesized that the reaction by naturalimmunity normally occurs for elimination of a specific cancer antigenderived from cancer cells and that then a specific immune response isinduced to cause the reaction of elimination of cancer cells bylymphocytes and other cells.

The recognition of a cancer cell-derived antigen requires the formationof a complex by a human leukocyte antigen (HLA) present on the cellsurface and a lymphocyte. The HLA molecule as a major histocompatibilityantigen is roughly divided into class I molecules (HLA types A, B, andC) and class II molecules (HLA types DP, DQ, and DR). The reaction ofelimination of a cancer cell by a cytotoxic T cell (CTL) is induced bythe specific recognition of a cancer antigen (CTL epitope) consisting of8 to 11 amino acids which is presented on an HLA class I molecule on thecancer cell surface by a T cell antigen receptor (TCR) on the CTL.

The search for immunogenic peptides has been currently carried out witha view to their application to the treatment or prevention of variousimmune-related diseases; for example, Japanese Patent Laid-Open No.08-151396 discloses that an oligopeptide consisting of a particularamino acid sequence has a HLA-binding capacity.

CITATION LIST Patent Literature

Patent Literature 1: Japanese Patent Laid-Open No. 08-151396

SUMMARY OF INVENTION Technical Problem

Many peptides having an HLA-binding capacity are known; however, thereis further a need for peptides capable of being used for the treatmentor prevention of various cancers. Since HLA gene is rich inpolymorphism, there is also a need for multi-type immunogenic peptideseach adaptable to a plurality of HLA types.

Solution to Problem

In view of the above-described circumstances, the present invention hasan object of providing an immunogenic peptide capable of binding to anHLA class I molecule, particularly a peptide capable of inducing CTL, apharmaceutical composition for treating or preventing cancer using thepeptide, an immunity inducer, and a method for producing anantigen-presenting cell.

Specifically, the present invention includes the following inventions.

(1) A peptide comprising 8 or more consecutive amino acid residues in anamino acid sequence of any of SEQ ID NOS: 1 to 11 and consisting of 11or less amino acid residues.

(2) The peptide according to (1), wherein in the amino acid sequence, 1or several amino acids are substituted, inserted, deleted, or added, andthe peptide has immunogenicity.

(3) The peptide according to (2), wherein in the amino acid sequence,the amino acid at position 2 is substituted by tyrosine, phenylalanine,methionine, tryptophan, valine, leucine, or glutamine, and/or the aminoacid at the C-terminal is substituted by phenylalanine, leucine,isoleucine, tryptophan, methionine, or valine.

(4) A pharmaceutical composition for treating or preventing cancer,comprising the peptide according to any one of (1) to (3).

(5) The pharmaceutical composition according to (4), wherein thecomposition is in the form of a vaccine.

(6) The pharmaceutical composition according to (4) or (5), wherein thepeptide can bind to one or more types of HLA molecules.

(7) An immunity inducer, comprising the peptide according to any one of(1) to (3).

(8) The immunity inducer according to (7), wherein the inducer is forinducing a cytotoxic T cell.

(9) The immunity inducer according to (7) or (8), wherein the peptidecan bind to one or more types of HLA molecules.

(10) A method for producing an antigen-presenting cell having aCTL-inducing activity, comprising a step of contacting the peptideaccording to any one of (1) to (3) with an antigen-presenting cell invitro.

Advantageous Effects of Invention

Attention has been given in recent years to immunotherapy as a methodfor treating cancer. The peptide of the present invention is stronglyexpected to have usefulness as a cancer vaccine because of its highHLA-binding capacity and also its high CTL-inducing capability. Itsapplications to various immunotherapies, particularly dendritic celltherapy, are also envisioned.

Glypican-3 (GPC3) is a protein belonging to the Glypican family.Glypican is one of proteoglycans and is known to bind toglycosyl-phosphatidylinositol on the cell surface. Glypican controls theactivity of various cell growth factors including Wnts, and the actionis believed to be due to acceleration or inhibition of interactionsbetween the cell growth factors and the receptors by Glypican. Inparticular, it has been revealed that GPC3 is expressed in almost allcases of hepatocellular carcinoma (HCC), and, on the other hand, ishardly expressed in normal liver, hepatic cirrhosis, and so on. Further,GPC3 is known to be highly expressed, not only in hepatocellularcarcinoma, but also in melanoma, ovarian cancer, and so on.

-   1. “Glypican-3: a marker and a therapeutic target in hepatocellular    carcinoma.” Jorge Filmus and Mariana Capurro, FEBS J., 280:    2471-2476, 2013.-   2. “Glypican-3: a new target for cancer immunotherapy.” Mitchell Ho    and Heungnam Kim, Eur. J. Cancer, 47, 333-338, 2011.

For hepatocellular carcinoma, clinical researches for a cancer vaccinewith a GPC3-derived peptide which is highly expressed in hepatocellularcarcinoma cells have been previously conducted, and the safety andimmunity-inducing ability have been reported.

-   3. “Peptide vaccines for hepatocellular carcinoma.” Daisuke Nobuoka,    Toshiaki Yoshikawa, Yu Sawada, Toshiyoshi Fujiwara and Tetsuya    Nakatsura, Human Vaccines & Immunotherapeutics, 9, 210-212, 2013.-   4. “Phase I Trial of a Glypican-3-Derived Peptide Vaccine for    Advanced Hepatocellular Carcinoma: Immunologic Evidence and    Potential of Improving Overall Survival.” Yu Sawada, et. al., Clin.    Cancer. Res., 18, 3636-3696, 2012.

In the present invention, several peptides have been identified each ofwhich is a GPC3-derived peptide different from any of the peptidesreported in the clinical researches, and binds to an HLA molecule andhas an immunity-inducing ability. Among the peptides of the presentinvention, a particular peptide can bind to a plurality of HLA types.Thus, the peptide of the present invention enables, for example, theprovision of a cancer vaccine and dendritic cell therapy covering anextremely wide range of cancer patients.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the results of ELISPOT assay (the number ofIFN-γ-producing cells) when samples derived from patients [0] (HLA type:24: 02/24:02) having received HSP70 dendritic cell therapy werestimulated with the peptide of SEQ ID NO: 1.

FIG. 2 is a graph showing the results of ELISPOT assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 02:01/24:02) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

FIG. 3 is a graph showing the results of ELISPOT assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 02:01/33:03) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

FIG. 4 is a graph showing the results of ELISA assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 24:02/26:01) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

FIG. 5 is a graph showing the results of ELISA assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 24:02/24:02) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

FIG. 6 is a graph showing the results of ELISA assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 11:01/24:02) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

FIG. 7 is a graph showing the results of ELISA assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 02:01/24:02) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

FIG. 8 is a graph showing the results of ELISA assay (the number ofIFN-γ-producing cells) when samples derived from patients (HLA type: 02:01/33:03) having received HSP70 dendritic cell therapy were stimulatedwith the peptide of SEQ ID NO: 1, 2, or 4.

DESCRIPTION OF EMBODIMENTS 1. Immunogenic Peptide

The peptides according to the present invention are each a peptidecomprising 8 or more consecutive amino acid residues in an amino acidsequence of any of SEQ ID NOS: 1 to 11 and consisting of 11 or less,preferably 10 or less, more preferably 9 or less amino acid residues intotal. The peptide of the present invention may be a peptide consistingof an amino acid sequence of any of SEQ ID NOS: 1 to 11. The peptide ofthe present invention is derived from GPC3, which is one of Glypican. Anamino acid sequence whose binding capacity to the HLA molecule is 3 ormore in terms of a −log Kd value has been selected, and the bindingcapacity here was predicted by the hypothesis obtained using an activelearning experiment method (Japanese Patent Laid-Open No. 08-151396)based on the amino acid sequence constituting GPC3.

The amino acid sequence constituting each peptide of the presentinvention and its HLA-binding prediction score are shown in Table 1below.

TABLE 1 Amino Acid Sequence Position Binding Prediction Score(SEQ ID NO:) in GPC3 To A*24:02 To A*02:01 To A*02:06 MVNELFDSL 1664.9889 4.5125 4.914 (SEQ ID NO: 1) LFDSLFPVI 170 5.3875 4.6623 4.8693(SEQ ID NO: 2) SALDINECL 190 5.0802 5.1752 4.9393 (SEQ ID NO: 3)SLQVTRIFL 222 4.9563 4.7937 4.9102 (SEQ ID NO: 4) SLTPQAFEF 136 5.36114.4718 4.5358 (SEQ ID NO: 5) GYICSHSPV 407 5.2631 4.5208 4.6902(SEQ ID NO: 6) ALNLGIEVI 232 4.7512 4.8916 4.8959 (SEQ ID NO: 7)LLQSASMEL  92 4.6163 4.9287 5.0213 (SEQ ID NO: 8) KLTTTIGKL 340 5.65264.2118 4.2929 (SEQ ID NO: 9) GMIKVKNQL 512 5.5052 4.3292 4.0995(SEQ ID NO: 10) ARLNMEQLL  85 5.0076 4.1008 4.2227 (SEQ ID NO: 11)

The peptide of the present invention has an HLA-binding capacity and hasimmunogenicity (hereinafter sometimes simply referred to as “HLApeptide” or “immunogenic peptide”). As used herein, “immunogenicity”means the ability to induce an immune response and, for example, meanshaving a CTL-inducing activity and consequently having a cytotoxicactivity against cancer cells.

In a preferred embodiment, the peptide of the present invention is amulti-HLA peptide capable of binding to a plurality of allelotypes ofHLA-A gene A. For example, the peptide of SEQ ID NO: 7 strongly binds toa product of HLA-A*24:02 gene (an HLA-A*24:02 molecule), a product ofHLA-A*02:01 gene (an HLA-A*02:01 molecule), and a product of HLA-A*02:06gene (an HLA-A*02:06 molecule), and has high immunogenicity.

The HLA subtype to which the peptide of the present invention can bindis not limited to HLA-A*24:02, HLA-A*02:01, or HLA-A*02:06. However,these HLA subtypes cover the order of 85% of oriental people includingthe Japanese and on the order of 55% of western people; thus, it isconsidered that the multi-HLA peptide of the present invention achievesa broad patient coverage, for example, in immunotherapy.

The peptide of the present invention may be modified in the amino acidresidues constituting the amino acid sequence of any of SEQ ID NOS: 1 to11 or a part thereof as long as it retains immunogenicity. The aminoacid sequence of each of SEQ ID NOS: 1 to 11 intends a state which ispresented on an antigen-presenting cell; however, when the peptide ofthe present invention is directly administered into the body, thepeptide sometimes experiences changes, such as the digestion of itsterminal in digestive organs and the like, depending on theadministration route. Thus, before incorporation into anantigen-presenting cell, the peptide of the present invention may bepresent in the form of a precursor which is formed by adding one or moreamino acid residues or the like at the N-terminal and/or C-terminal sothat amino acid sequence of any of SEQ ID NOS: 1 to 11 are retained uponbinding to a predetermined HLA class I molecule on theantigen-presenting cell.

In addition, the peptide of the present invention may have 1 or severalamino acid residues constituting the peptide of the present inventionsubstituted, inserted, deleted, or added, and/or have modifications,such as sugar chain addition, side chain oxidation, and/orphosphorylation, as long as the peptide has desired immunogenicity.“Amino acid” herein is used in its most comprehensive sense and includesartificial amino acid variants and derivatives in addition to naturalamino acids. Examples of the amino acid herein include natural proteinL-amino acids; D-amino acids; chemically modified amino acids, such asamino acid variants and derivatives; natural non-protein amino acids,such as norleucine, β-alanine, and ornithine; and chemically synthesizedcompounds having properties known in the art, characteristic of aminoacids. Examples of the non-natural amino acid include α-methyl aminoacids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids(e.g., β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, andα-methyl-histidine), amino acids having extra methylene on the sidechain (“homo” amino acids), and amino acids in each of which thecarboxylic acid functional group on the side chain is substituted by asulfonic acid group (e.g., cysteic acid).

For the substitution of an amino acid residue, and the like, inconsideration of the regularity of a peptide sequence having a bindingcapacity to HLA (J. Immunol., 152: p3913, 1994; Immunogenetics, 41:p178, 1995; J. Immunol., 155: p4307, 1994), those skilled in the art canproperly substitute an amino acid residue as a constituent of thepeptide of the present invention.

More specifically, in the case of a peptide binding to an HLA-A*24:02molecule, the amino acid at position 2 of the peptide may be substitutedby tyrosine, phenylalanine, methionine, or tryptophan, and/or theC-terminal amino acid may be substituted by phenylalanine, leucine,isoleucine, tryptophan, or methionine. In the case of a peptide bindingto an HLA-A*02:01 molecule, the amino acid at position 2 may besubstituted by leucine or methionine, and/or the C-terminal amino acidmay be substituted by valine or leucine. In addition, in the case of apeptide binding to an HLA-A*02:06 molecule, the amino acid at position 2may be substituted by valine or glutamine, and/or the C-terminal aminoacid may be substituted by valine or leucine.

Each peptide of the present invention can be produced using a techniqueknown to those skilled in the art. For example, it may be artificiallysynthesized by a solid-phase method, such as the Fmoc method or the tBocmethod, or a liquid-phase method. A desired peptide may also be producedby expressing a polynucleotide encoding the peptide of the presentinvention or a recombinant vector containing the polynucleotide. Thepeptides thus obtained can each be identified using a technique known tothose skilled in the art. For example, it can be identified using theEdman degradation method or a mass spectrometry method.

2. Pharmaceutical Composition

The pharmaceutical composition for treating or preventing canceraccording to the present invention contains, as an active ingredient,for example, a peptide containing 8 or more consecutive amino acidresidues in one or more amino acid sequences selected from the groupconsisting of SEQ ID NOS: 1 to 11 and consisting of 11 or less,preferably 10 or less, more preferably 9 or less amino acid residues intotal. The peptide contained in the pharmaceutical composition may be apeptide consisting of an amino acid sequence of any of SEQ ID NOS: 1 to11. The peptide is as defined hereinbefore.

The peptide of the present invention induces CTL by being presented onan antigen-presenting cell, and the induced CTL injures a cancer cell.Thus, the active ingredient of the pharmaceutical composition of thepresent invention is not limited to the peptide of the presentinvention, and may be a component capable of directly or indirectlyinducing CTL, for example, a polynucleotide encoding the peptide or avector containing the polynucleotide, or an antigen-presenting cellpresenting a complex of the peptide and an HLA molecule on the surfaceor an exosome secreted from the antigen-presenting cell, or acombination thereof. Examples of the antigen-presenting cell usedinclude a macrophage and a dendritic cell; however, it is preferable touse the dendritic cell, which has a high CTL-inducing capability. Any ofother ingredients known to be used for cancer therapy, such as achemokine, a cytokine, a tumor necrosis factor, and a chemotherapeuticagent, may be contained in the pharmaceutical composition of the presentinvention. The dose of the peptide may be, for example, about 1 to 10 mgper day when the patient is an adult. However, the dose varies dependingon the age and body weight of the patient, the administration method,and the like, and thus is properly determined by those skilled in theart.

The pharmaceutical composition of the present invention is thought to beuseful for the killing of cancer cells by, for example, but not intendedto be limited to, the following action mechanism. The administration ofthe pharmaceutical composition of the present invention to a particularcancer patient results in that the peptide in the pharmaceuticalcomposition is presented in a state in which it is bound to an HLAmolecule on the antigen-presenting cell surface. On recognizing thepeptide on such an antigen-presenting cell, CTL is activated,proliferated, and systemically circulated. When the peptide-specific CTLenters cancer tissue, it recognizes the same peptide derived from aspecific cancer antigen, naturally binding to an HLA molecule present onthe cancer cell surface to kill the cancer cell. Such an actioncontributes to the cancer treatment.

The pharmaceutical composition of the present invention can be used notonly for treating cancer but also for preventing cancer. For example,the administration of the pharmaceutical composition of the presentinvention into a healthy human body induces CTL, and the inducedcytotoxic T cell stay in the body and thus, when a particular cancercell occurs, can injure the cancer cell. Similarly, the composition maybe administered into a human body after treating cancer to prevent therecurrence of the cancer.

Any cancer expressing GPC3 is contemplated as a cancer to be treated orprevented. More specific examples of the cancer of interest include, butnot intended to be limited to, hepatocellular cancer, cutaneous cancersuch as melanoma, and ovarian cancer. For example, since GPC3 from whichthe peptide of the present invention is derived is overexpressed inhepatocellular cancer, it is considered that the peptide of the presentinvention is effective particularly in treating or preventing thehepatocellular cancer. When a plurality of cancers to be treated orprevented are present, a plurality of active ingredients, including theimmunogenic peptide, may be contained in the pharmaceutical compositionof the present invention.

The pharmaceutical composition of the present invention can be dissolvedin an aqueous solvent, formulated in the form of a pharmaceuticallyacceptable salt, and administered to patients. Examples of the form ofsuch a pharmaceutically acceptable salt include a form buffered atphysiological PH in the form of a physiologically acceptablewater-soluble salt, for example, a salt of sodium, potassium, magnesium,or calcium. In addition to the water-soluble solvent, anon-water-soluble solvent may also be used; examples of such anon-water-soluble solvent include alcohols, such as ethanol andpropylene glycol.

The formulation containing the pharmaceutical composition of the presentembodiment may contain agents for various purposes; examples of suchagents include a preservative and a buffer agent. Examples of thepreservative include sodium bisulfite, sodium bisulfate, sodiumthiosulfate, benzalkonium chloride, chlorobutanol, thimerosal,phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinylalcohol, phenylethyl alcohol, ammonia, dithiothreitol, andbeta-mercaptoethanol. Examples of the buffer agent include sodiumcarbonate, sodium borate, sodium phosphate, sodium acetate, and sodiumbicarbonate. These agents can be present in an amount capable ofmaintaining the pH of a system at 2 to 9, preferably 4 to 8.

The dosage form of the pharmaceutical composition of the presentinvention is not particularly limited; however, when it is used in theform of a vaccine, examples of its dosage form include injections(intramuscular, subcutaneous, and intracutaneous), oral formulations,and nasal drop formulations. When the pharmaceutical composition of thepresent invention is in the form of a vaccine, it may be a mixedcocktail vaccine containing a plurality of active ingredients. Forexample, such a vaccine can contain any two or more of the peptides ofSEQ ID NOS: 1 to 11, or contain a plurality of active ingredients bycombination with other active ingredients.

The vaccine of the present invention may be an inertingredient-containing vaccine containing an ingredient which is aningredient other than the pharmaceutical composition, has no activityper se, and has the effect of further enhancing the effect of thepharmaceutical composition as a vaccine. Examples of the inertingredient include an adjuvant and a toxoid. Examples of the adjuvantinclude, but not intended to be limited to, precipitation type ones,such as aluminium hydroxide, aluminium phosphate, and calcium phosphate,and oily type ones, such as Freund's complete adjuvant and Freund'sincomplete adjuvant.

When present in the form of a vaccine, the pharmaceutical composition ofthe present invention is preferably administered into the body byinjection or infusion, such as intracutaneous, subcutaneous, orintramuscular administration, or by dermal administration or inhalationthrough the mucosa of the nose, pharynx, or the like. Its single dosecan be set to between a dose capable of significantly inducing cytotoxicT cells and a dose at which a significant number of non-cancer cellsexperience injury.

The pharmaceutical composition of the present invention is contemplatedfor not only administration to a human body but also extracorporeal use.More specifically, the pharmaceutical composition of the presentinvention may be used for the purpose of stimulating anantigen-presenting cell in vitro or ex vivo to increase its CTL-inducingactivity. For example, in a case where the pharmaceutical composition ofthe present invention is used for dendritic cell therapy for cancer, thecomposition can be contacted with antigen-presenting cells, such asdendritic cells, derived from a patient in need of cancer treatment orprevention in advance, followed by administering the antigen-presentingcells to the patient by returning them into the patient's body. Thepeptide contained in the pharmaceutical composition can be introducedinto an antigen-presenting cell, for example, by a lipofection method oran injection method. When a polynucleotide encoding the peptide of thepresent invention is used in such an application, the polynucleotide canbe introduced into an antigen-presenting cell by a technique known inthe art. For example, an antigen-presenting cell derived from a patientmay be transformed in vitro using a polynucleotide of interest or avector encoding the polynucleotide by a lipofection method, anelectroporation method, a microinjection method, a cell fusion method, aDEAE dextran method, a calcium phosphate method, or the like.

3. Immunity Inducer

The immunity inducer according to the present invention contains, as anactive ingredient, for example, a peptide containing 8 or moreconsecutive amino acid residues in one or more amino acid sequencesselected from the group consisting of SEQ ID NOS: 1 to 11, andconsisting of 11 or less, preferably 10 or less, more preferably 9 orless amino acid residues. The peptide contained in the immunity inducermay be a peptide consisting of an amino acid sequence of any of SEQ IDNOS: 1 to 11. The peptide is as defined hereinbefore.

It is considered that the peptide of the present invention inducesimmunity by being presented on an antigen-presenting cell. Thus, theactive ingredient of the immunity inducer of the present invention isnot limited to the peptide of the present invention, and may be acomponent capable of directly or indirectly inducing immunity, forexample, a polynucleotide encoding the peptide of the present inventionor an expression vector containing the peptide, or an antigen-presentingcell presenting a complex of the peptide and an HLA molecule on thesurface or an exosome secreted from the antigen-presenting cell, or acombination thereof. Examples of the antigen-presenting cell usedinclude a macrophage and a dendritic cell; however, it is preferable touse the dendritic cell, which has a high CTL-inducing capability.

The immunity inducer of the present invention is contemplated for notonly administration to a human body but also extracorporeal use. Morespecifically, the immunity inducer of the present invention may be usedfor the purpose of stimulating an antigen-presenting cell in vitro or exvivo to increase its CTL-inducing activity. For example, in a case wherethe immunity inducer of the present invention is used for dendritic celltherapy, the inducer can be contacted with antigen-presenting cells,such as dendritic cells, derived from a patient in need of immunityinduction in advance, followed by administering the antigen-presentingcells to the patient by returning them into the patient's body. Thepeptide contained in the immunity inducer can be introduced into anantigen-presenting cell, for example, by transfection via a liposome (alipofection method) or an injection method. When a polynucleotideencoding the peptide of the present invention is used in such anapplication, the polynucleotide can be introduced into anantigen-presenting cell by a technique known in the art. For example, anantigen-presenting cell derived from a patient may be transformed invitro using a polynucleotide of interest or a vector expressing thepolynucleotide by a lipofection method, an electroporation method, amicroinjection method, a cell fusion method, a DEAE dextran method, acalcium phosphate method, or the like.

As used herein, “immunity induction” means inducing an immune response,for example, increasing the CTL-inducing activity of anantigen-presenting cell, and further increasing the cytotoxic activityof CTL against a cancer cell. As used herein, “CTL induction” meansinducing or proliferating CTL specifically recognizing a certainantigen, or differentiating a naive T cell into an effector cell havingthe ability to kill a target cell (cytotoxic activity), such as a cancercell, and/or increasing the cytotoxic activity of CTL by thepresentation of the peptide of the present invention on theantigen-presenting cell surface in vitro or in vivo. The CTL-inducingactivity can be measured by evaluating the production of cytokines (forexample, interferon (IFN)-γ)) by CTL. For example, the CTL-inducingactivity may be measured by evaluating an increase in cytokine-producingcells induced from precursor cells by antigen-presenting cells, such asperipheral-blood monocytes, stimulated with the peptide of the presentinvention, using a known high-sensitive immunoassay, such as ELISPOT(Enzyme-Linked ImmunoSpot) assay and ELISA (Enzyme-Linked ImmunoSorbentAssay). The cytotoxic activity can also be measured by a known method,such as a ⁵¹Cr release method. When the activity is significantlyincreased, for example, by 5% or more, 10% or more, 20% or more,preferably 50% or more, compared to control, immunity or CTL can beevaluated to have been induced.

4. Method for Producing Antigen-Presenting Cell

The method for producing an antigen-presenting cell according to thepresent invention includes a step of contacting, for example, a peptidecontaining 8 or more consecutive amino acid residues in one or moreamino acid sequences selected from the group consisting of SEQ ID NOS: 1to 11, and consisting of 11 or less, preferably 10 or less, morepreferably 9 or less amino acid residues in total, with anantigen-presenting cell in vitro. The peptide used in the productionmethod of the present invention may be a peptide consisting of an aminoacid sequence of any of SEQ ID NOS: 1 to 11. The peptide is as definedhereinbefore.

It is considered that the peptide used in the production method of thepresent invention binds to an HLA class I molecule on theantigen-presenting cell surface, is presented to CTL as an antigenpeptide, and thereby induces the CTL activity of the antigen-presentingcell. Thus, the component to be contacted with an antigen-presentingcell is not limited to the peptide of the present invention, and may bea component capable of directly or indirectly inducing CTL, for example,a polynucleotide encoding the peptide or a vector containing thepolynucleotide, or an antigen-presenting cell presenting a complex ofthe peptide and an HLA molecule on the surface or an exosome secretedfrom the antigen-presenting cell, or a combination thereof. Examples ofthe antigen-presenting cell used include a macrophage and a dendriticcell; however, it is preferable to use the dendritic cell, which has ahigh CTL-inducing capability.

The antigen-presenting cell produced by the production method of thepresent invention is contemplated to be not only used as an activeingredient of the pharmaceutical composition or the immunity inducer butalso used for immunotherapy and the like. For example, in a case wherethe antigen-presenting cells produced are used for dendritic celltherapy for cancer, the cells can be contacted with antigen-presentingcells, such as dendritic cells, having a low CTL-inducing capability,derived from a patient in need of immunity induction in advance,followed by administering the antigen-presenting cells to the patient byreturning them into the patient's body. The peptide of the presentinvention can be introduced into an antigen-presenting cell, forexample, by transfection via a liposome (a lipofection method) or aninjection method. When a polynucleotide encoding the peptide of thepresent invention is used in such an application, the polynucleotide canbe introduced into an antigen-presenting cell by a technique known inthe art. For example, an antigen-presenting cell derived from a patientmay be transformed in vitro using a polynucleotide of interest or avector encoding the polynucleotide by a lipofection method, anelectroporation method, a microinjection method, a cell fusion method, aDEAE dextran method, a calcium phosphate method, or the like.

Example 1

The present invention will be more specifically described below withreference to Examples. However, the present invention is not intended tobe limited thereto.

Specifically, the procedures of prediction, experiment, and evaluationin this Example were carried out based on the active learning experimentdesign described in International Publication No. WO 2006/004182. A rulewas constructed by repeating the following steps as a whole.

(1) A low rank learning algorithm to be described hereinafter is oncetried. That is, a plurality of hypotheses are generated based on randomresampling from accumulated data, and the point is chosen at which thevariance of predicted values of randomly generated candidate querypoints (peptides) is largest as the query point to be experimented.

(2) The peptide at the chosen query point is produced by synthesis andpurification methods to be described hereinafter. The actual bindingcapacity is measured by an experiment to be described hereinafter, andadded to the accumulated data.

Performing such an active learning method could reduce the number ofbinding experiments which are otherwise necessary to carry out for allof 5 hundred billion (=20⁹) or more candidate substances of HLA-bindingpeptides consisting of 9 amino acid residues.

Using the rule as described above, the amino acid sequences of SEQ IDNOS: 1 to 11 were extracted.

<Synthesis and Purification of Peptide>

The peptides having the amino acid sequences of SEQ ID NOS: 1 to 11 weremanually synthesized by the Merrifield solid-phase method using Fmocamino acids. The resultant were deprotected and then subjected toreverse-phase HPLC purification using a C18 column to a purity of 95% ormore. The identification of the peptides and confirmation of the puritythereof were performed by MALDI-TOF mass spectrometry (AB SCIEXMALDI-TOF/T0F5800). Peptide quantification was carried out by Micro BCAassay (Thermo Scientific Co., Ltd.) using BSA as a standard protein.

<Binding Experiment of Peptide to HLA-A*24:02 Molecule>

The binding capacity of each peptide to the HLA-A*24:02 molecule as theproduct of HLA-A*24:02 gene was measured using C1R-A24 cells expressingthe HLA-A*24:02 molecule (the cells prepared by Prof. MasafumiTakeguchi, Kumamoto University were gifted by Assoc. Prof. MasakiYasukawa, Ehime University with permission).

First, C1R-A24 cells were exposed to acidic conditions of pH 3.3 for 30seconds to dissociate and remove endogenous peptides which wereoriginally bound to the HLA-A*24:02 molecule and a light chain, β2m,which was commonly associated with HLA class I molecules. Afterneutralization, purified β2m was added to the C1R-A24 cells, which wasthen added to peptide dilution series. The mixtures were each thenincubated on ice for 4 hours. The 3-molecule assembly (MHC-pep)consisting of the HLA-A*24:02 molecule, the peptide, and β2 m which hadbeen reasociated during the incubation was stained with a fluorescentlabeled monoclonal antibody, 17A12, recognizing the assembly.

Subsequently, the number of MHC-pep's per C1R-A24 cell (which isproportional to the fluorescent intensity of the above fluorescentantibody) was quantitatively measured using a fluorescent cell analyzer,FACScan (Becton, Dickinson and Company). The binding dissociationconstant, Kd value, between the HLA-A*24:02 molecule and the peptide wascalculated from the average fluorescent intensity per cell using amethod as published in a paper (Udaka et al., Immunogenetics, 51,816-828, 2000) by the present inventor.

<Binding Experiment of Peptide to HLA-A*02:01 Molecule>

The binding capacity of each peptide to the HLA-A*02:01 molecule as theproduct of HLA-A*02:01 gene was measured using a cell line, T2,(purchased from ATCC) expressing the HLA-A*02:01 molecule.

T2 cells and purified β2m were added to stepwise dilution series of apeptide whose binding capacity was to be measured, which was thenincubated at 37° C. for 4 hours. The HLA-A*02:01 molecule whoseexpression level was concentration-dependently increased by this timepoint was stained with an assembly-specific fluorescent labeledmonoclonal antibody, BB7.2.

Thereafter, the amount of fluorescence per cell was measured using aflow cytometer, and the dissociation constant, Kd value, was calculatedusing a method as published in a paper by the present inventor (Udaka etal., Immunogenetics, 51, 816-828, 2000).

<Binding Experiment of Peptide to HLA-A*02:06 Molecule>

The binding capacity of each peptide to the HLA-A*02:06 molecule as theproduct of HLA-A*02:06 gene was measured using RA2.6 cells (a cell linenewly prepared at Kochi University) in which cDNA of the HLA-A*02:06gene was introduced into RMAS as a mouse TAP (transporter associatedwith antigen processing)-deficient cell line.

First, the RA2.6 cells were cultured overnight at 26° C. to accumulatethe HLA-A*02:06 molecules unbound to the peptide on the cell surface.Any of peptide dilution series was added thereto for binding at 26° C.for 60 minutes.

Subsequently, the mixture was cultured at 35° C. for 4 hours, resultingin the denaturation of the empty HLA-A*02:06 molecule unbound to thepeptide and the loss of its steric structure. A fluorescent labeledmonoclonal antibody, BB7.2, specifically recognizing a peptide-boundHLA-A*02:06 molecule, was added thereto, which was then incubated on icefor 20 minutes to stain the cells.

Thereafter, the amount of fluorescence per cell was measured using aflow cytometer, and the dissociation constant, Kd value, was calculatedusing a method as published in a paper by the present inventor (Udaka etal., Immunogenetics, 51, 816-828, 2000).

<Evaluation Result of Binding Experiment>

As a result, the binding experiment data of the peptides of the presentinvention to each HLA molecule as shown in the following table wereobtained.

TABLE 2 Amino Acid Sequence Position Binding Experiment Data(SEQ ID NO:) in GPC3 To A*24:02 To A*02:01 To A*02:06 MVNELFDSL 166 -4.699595957  -5.305836823  -6.140220372 (SEQ ID NO: 1) LFDSLFPVI 170 -7.427547379  -5.06520602 >-3 (SEQ ID NO: 2) SALDINECL 190 >-3 >-3 -6.316321482 (SEQ ID NO: 3) SLQVTRIFL 222  -5.336368455  -6.210154227 -5.213788842 (SEQ ID NO: 4) SLTPQAFEF 136  -7.113561896  -4.9256496 -4.515516445 (SEQ ID NO: 5) GYICSHSPV 407  -6.548090575 >-3 >-3(SEQ ID NO: 6) ALNLGIEVI 232  -3.956124763  -5.917879215  -4.161231756(SEQ ID NO: 7) LLQSASMEL  92  -5.48762276  -6.136635191  -5.877071673(SEQ ID NO: 8) KLTTTIGKL 340  -5.211802039  -4.970696549  -5.033936719(SEQ ID NO: 9) GMIKVKNQL 512  -6.904149343  -4.757453097  -3.357850496(SEQ ID NO: 10) ARLNMEQLL  85  -5.203320264  -3.84036042  -2.764152073(SEQ ID NO: 11)

The amino acid sequences of SEQ ID NOS: 1 to 11 are derived from thefull-length sequence of the predetermined genomic protein of GPC3registered in GENBANK (SEQ ID NO: 12) (>gi|4758462|ref|NP_004475.1|glypican-3 isoform 2 precursor [Homo sapiens]).

<Immunity Induction Test of Peptide>

(1) Preparation of Peptide-Stimulated Dendritic Cell

Day 0 to 9 (Induction of Dendritic Cell)

Of peripheral blood monocytes obtained by pheresis from the patient [0]treated with HSP70 dendritic cell therapy, a cell fraction adhering tothe culture flask was cultured in AIM-CM medium (trade name “Gibco” fromThermo Fisher Scientific Co., Ltd.) at 37° C. for 10 days. Duringculture, 15 μl of IL-4 and 30 μl of granulocyte-monocytecolony-stimulating factor (GM-CSF) were added to the medium at day 0 andday 3, and 15 μl of IL-4, 30 μl of GM-CSF, and 75 μl of tumor necrosisfactor (TNF)-α were added at day 5.

Day 10 (Stimulation with Peptide and Recovery of Dendritic Cell)

The dendritic cells induced were newly recovered into AIM-CM medium, andthe peptides of the present invention (SEQ ID NOS: 1 to 11) were eachadded to 20 μg/ml. Then, the medium containing the dendritic cells wascultured at 37° C. for 2 hours. The following peptides were used aspositive and negative controls.

Positive control for HLA-A*24:02 (EBV LMP2, 419-427: TYGPVFMCL (SEQ IDNO: 13))

Negative control for HLA-A*24:02 (HIV env gp160, 584-592: RYLRDQQLL (SEQID NO: 14))

Positive control for HLA-A*02:01 (Flu A MP, 58-66: GILGFVFTL (SEQ ID NO:15))

Negative control for HLA-A*02:01 (HIV gap p17, 77-85: SLYNTVATL (SEQ IDNO: 16))

Positive control for HLA-A*02:06 (EBV LMP2 453-461: LTAGFLIFL (SEQ IDNO: 17))

Negative control for HLA-A*02:06 (HIV gap p24 341-349: ATLEEMMTA (SEQ IDNO: 18))

The dendritic cells were recovered, washed 3 times or more with asufficient amount of AIM-CM medium, and counted.

(2) Preparation of CD8T Cell

Day 0 to 9

Of peripheral blood monocytes obtained by pheresis from the patienttreated 2 times or more with the above vaccine, a floating cell fraction(including lymphocytes) not adhering to the culture flask was culturedin AIM-CM medium (from GIBCO Co., Ltd.) at 37° C. for 10 days. Duringculture, 40 μl of IL-2 was added to the medium at day 4 and day 6.

Day 10

Using CD8 Negative Selection Kit (from Miltenyi Biotec), CD8T cells wereseparated from the medium and counted.

(3) Coculture

The dendritic cells and the CD8T cells obtained in (1) and (2) abovewere cocultured in AIM medium at 37° C. under the following conditions.

-   -   CD8T cells: 5×10³ cells/well    -   Dendritic cells: 2×10³ cells/well

Day 12 or 13

To the above medium was added 0.4 ml/well of AIM-CM medium containingIL-2 in an amount of 20 U/ml.

(4) ELISPOT Assay

Day 17

The CD8T cells were added to a 96-well plate for ELISPOT (fromMillipore), coated with an anti-TFN-γ monoclonal antibody (from MabtechAB) to 2×10⁴ cells/well. For each sample, 3 or more wells were used. Toeach well was added 100 μl of AIM-V (from trade name “Gibco” from ThermoFisher Scientific Co., Ltd.). The 96-well plate for ELISPOT was culturedat 37° C.

Day 18

The anti-TFN-γ antibody was added to each well and further reacted withan HRP enzyme-labeled secondary antibody to measure the number ofIFN-γ-producing cells by color reaction. As typical results of theELISPOT assay, those for patients whose HLA type was 24:02/24:02 areshown in FIG. 1; those for patients, 02:01/24:02, in FIG. 2; and thosefor patients, 02:01/33:03, in FIG. 3. In each figure, the average of 3assay results are indicated.

(5) ELISA Assay

Day 17

A culture supernatant at day 7 after coculture of T cells with dendriticcells pulsed with each of the above peptides was diluted to the 4 levelsof ×1, ×5, ×25, and ×125 to identify the dilution level falling withinthe limit of measurement using Human IFN-γ ELISA MAX Deluxe Set (fromBioLegend Inc.). Thereafter, each sample was measured 3 times at theidentified dilution level. As typical results of the ELISA assay, thosefor patients whose HLA type was 24:02/26:01, those for patients whoseHLA type was 24:02/24:02, those for patients whose HLA type was11:01/24:02, those for patients whose HLA type was 02:01/24:02, andthose for patients whose HLA type was 02:01/33:03, are shown in FIGS. 4to 8 respectively.

The present invention has been described above based on Example. ThisExample is merely illustrative, and it should be understood by thoseskilled in the art that various modifications may be made and that themodifications are also within the scope of the present invention.

1. A peptide comprising 8 or more consecutive amino acid residues but 11or less amino acid residues of SEQ ID NO:
 4. 2. A pharmaceuticalcomposition for treating or preventing cancer, comprising the peptideaccording to claim
 1. 3. The pharmaceutical composition according toclaim 2, wherein the composition is in the form of a vaccine.
 4. Thepharmaceutical composition according to claim 3, wherein the peptide canbind to one or more types of HLA molecules.
 5. An immunity inducer,comprising the peptide according to claim
 1. 6. The immunity induceraccording to claim 5, wherein the inducer is for inducing a cytotoxic Tcell.
 7. The immunity inducer according to claim 6, wherein the peptidecan bind to one or more types of HLA molecules.
 8. A method forproducing an antigen-presenting cell having CTL-inducing activity,comprising a step of contacting the peptide according to claim 1 with anantigen-presenting cell in vitro.
 9. A method for treating or preventingcancer in a subject, comprising administering to the subject a peptidecomprising 8 or more consecutive amino acid residues but 11 or lessamino acid residues of SEQ ID NO: 4.